Production of cheese and cultured dairy products have long relied on the fermentation of milk with lactococci (previously classified as the group N Streptococci), such as Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactis biovar. diacetylactis (also called Lactococcus diacetylactis). These bacteria are responsible for the acid development, flavor production, and often coagulum characteristics in mesophilic dairy fermentations. Because efficient milk fermentations are dependent on the growth and activity of the lactococci, care is exercised to prepare bacterial starter cultures that are highly active and uncontaminated with undesirable microorganisms or bacteriophages. However, the fermentation process itself is nonaseptic, occurring in open vats with a nonsterile medium (pasteurized milk). For the majority of strains of lactococci employed in commercial dairy fermentations, lytic bacteriophages capable of halting growth and acid production can appear within one to two days after introducing the starter culture into the cheese plant. Bacteriophage contamination of numerous industrial fermentations other than milk fermentations have also been observed. Lytic phages continue to disrupt fermentative activities and cause substantial economic losses, most notably in the cultured dairy product industries (See, e.g., T. Klaenhammer, Biochemical Society Transactions, 19:675(1991)).
The increased production capacity and process efficiency in the dairy industry in recent decades has necessitated the use of defined mixtures of lactococci capable of uniform and rapid rates of acid production. With the selection of highly fermentative lactococci and their propagation under aseptic conditions (in the absence of bacteriophages), the majority of cultures now used by the industry are highly susceptible to bacteriophage attack upon introduction into the factory environment. A number of methods have been developed to minimize phage action during commercial milk fermentations, including the use of concentrated cultures, aseptic bulk starter vessels and phage-inhibitory media (see, e.g., U.S. Pat. No. 4,282,255). However, phage contamination cannot be prevented following entrance into the fermentation vat. Emphasis for protection of the culture therefore has shifted to minimizing prolific phage-host interactions through rotation of phage-unrelated strains or the use of phage-resistant mutants in multiple-strain starters. Although in theory strain rotation should minimize developing phage populations within the plant, in practice it has been shown that rotations of large numbers of strains can lead to an increase in phage populations and diversity within a cheese plant (See R. Thunell et al., J. Dairy Sci. 64, 2270-2277 (1981)).
Hershberger, U.S. Pat. No. 4,530,904, discloses a method for protecting bacteria in general from different types of bacteriophage. The method involves transforming a bacterium with a recombinant DNA cloning vector comprising a replicon that is functional in the bacterium, a gene that expresses a functional polypeptide (i.e., human growth hormone) in the bacterium, and a DNA segment which confers restriction and modification activity to the bacterium. The transformed bacterium is then cultured under large-scale fermentation conditions. This method is particularly adapted to fermentation procedures for the production of polypeptides such as growth hormone.
U.S. Pat. No. 4,732,859 to Hershberger et al. relates to a method of protecting various genera of bacteria from naturally occurring bacteriophage by providing host bacterial cells with a restriction system that digests HhaII site-containing foreign DNA (found in most naturally occurring phages) and renders the bacteriophage non-functional. Bacteria are transformed with a recombinant DNA cloning vector which comprises a gene that expresses a restriction endonuclease which confers restriction activity to the bacteria.
U.S. Pat. Nos. 4,918,014 and 4,874,616, both to Vedamuthu, are directed to a method of imparting bacteriophage resistance to bacteriophage sensitive strains of Lactococcus, whereby a plasmid encoding the production of a mucoid substance is conjugally transferred via a plasmid into a bacteriophage sensitive strain. Additional strategies used for the construction of bacteriophage-insensitive strains include the introduction of one or more resistance mechanisms within a single host, or introducing a single plasmid containing more than one resistance mechanism. For recent reviews, see Klaenhammer, T. R., FEMS Microbiol. Rev. 46:313-325 (1987); Sanders, Biochimie 70: 411-422 (1988); and C. Hill, FEMS Microbiol. Rev. 12, 87-108 (1993).
Plasmids and plasmid derivatives which confer bacteriophage restriction and modification (R/M) activity to lactococci containing the plasmid or derivative are described in U.S. Pat. Nos. 4,883,756, 4,931,396, 5,139,950, and 5,109,506.
Despite increased protection of starter cultures by rotation strategies or by use of phage resistant derivatives, new lytic bacteriophages appear routinely in industrial fermentations, disrupting the fermentative process and causing loss of substrates and products. Bacteriophages infecting Lactococcus strains have been classified into twelve distinct species (Jarvis, et al. Intervirology, 32:2(1991)). The lytic phages which have been the most prevalent in the dairy industry are classified in the species c2 and 936 and are composed of prolate- and small isometric-headed phages, respectively (Braun, et al. J. Gen. Microbiol. 135:2551(1992); Coveney et al., Appl. Environ. Microbiol. 53:1439(1987); Moineau, et al. Can. J. Microbiol. 38:875(1992)). More recently, another phage species, P335, has emerged with increasing frequencies in cheese plants (Alatossava and Klaenhammer. Appl. Environ. Microbiol. 57:1346(1991); Moineau, Pandian and Klaenhammer. Appl. Environ. Microbiol. 59:197(1993)).
The origin of new lactococcal bacteriophages in the dairy industry has been investigated, see, e.g., Davidson et al., FEMS Microbiol. Rev. 87:79 (1990); Jarvis, J. Dairy Sci., 72:3406(1989). The genetic events believed to contribute to the evolution of new phage strains are infrequent, complex, and in general remain to be elucidated.